Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The mortality rate is roughly that of cancer. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis.[unreadable] The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5' exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date there are nearly 150 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). Presently, there are nearly 150 pathogenic disease mutations in the POLG gene that cause PEO, ataxia-neuropathy and Alpers syndrome.[unreadable] [unreadable] Mutations in the POLG gene have emerged as one of the most common causes of inherited mitochondrial disease in children and adults. They are responsible for a heterogeneous group of at least 6 major phenotypes of neurodegenerative disease that include: 1) childhood Myocerebrohepatopathy Spectrum disorders (MCHS), 2) Alpers syndrome, 3) Ataxia Neuropathy Spectrum (ANS) disorders, 4) Myoclonus Epilepsy Myopathy Sensory Ataxia (MEMSA), 5) autosomal recessive Progressive External Ophthalmoplegia (arPEO), and 6) autosomal dominant Progressive External Ophthalmoplegia (adPEO). Due to the clinical heterogeneity, time-dependent evolution of symptoms, overlapping phenotypes, and inconsistencies in muscle pathology findings, definitive diagnosis relies on the molecular finding of deleterious mutations. In a multicenter North American collaboration involving 17 clinical sites research headed up by Baylor and NIEHS we analyzed the entire POLG DNA sequence from approximately 350 patients displaying a phenotype consistent with POLG related mitochondrial disease. Of these 350 patients, 61 (17%) had one or more mutant POLG alleles. Two mutant alleles were identified in 31 unrelated index patients with autosomal recessive POLG-related disorders. Among them, 20 (67%) had Alpers syndrome, 4 (13%) had arPEO, and 3 (10%) had ANS. In addition, 30 patients carrying one altered POLG allele were found. A total of 25 novel alterations were identified, including 6 null mutations. They concluded that sequence analysis allows the identification of mutations responsible for POLG-related disorders and, in most of the autosomal recessive cases where two mutant alleles are found in trans, finding deleterious mutations provides an unequivocal diagnosis of the disease.[unreadable] We described the clinical features, muscle pathological characteristics, and molecular studies of a 42-year-old patient experienced hearing loss, PEO, loss of central vision, macrocytic anemia, and hypogonadism with a mutation in the gene encoding the accessory subunit (p55) of polymerase gamma (POLG2) and a mutation in the OPA1 gene. A muscle biopsy specimen showed scattered intensely succinate dehydrogenase-positive and cytochrome-c oxidase-negative fibers. Southern blot of muscle mitochondrial DNA showed multiple deletions. The results of screening for mutations in the nuclear genes associated with PEO and multiple mitochondrial DNA deletions, including those in POLG, ANT1 (gene encoding adenine nucleotide translocator 1), and PEO1, were negative, but sequencing of POLG2 revealed a G1247C mutation in exon 7, resulting in the substitution of a highly conserved glycine with an alanine at codon 416 (G416A). Because biochemical analysis of the mutant protein showed no alteration in chromatographic properties and normal ability to protect the catalytic subunit from N-ethylmaleimide, we also sequenced the OPA1 gene and identified a novel heterozygous mutation (Y582C). Although we initially focused on the mutation in POLG2, the mutation in OPA1 is more likely to explain the late-onset PEO and multisystem disorder in this patient.[unreadable] We found a biochemical link that explains the enhanced oxidative stress and DNA mutagenesis that occurs in patients suffering from PEO with Parkinson symptoms. Previous work had demonstrated that the DNA polymerase gamma present in these patients carried the Y955C mutation that, in turn, induced replication stalling and DNA deletions. Because these patients also develop Parkinson symptoms, and animal models show enhanced incorporation of 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxo-dG), a common oxidative lesion into mitochondrial DNA, we wanted to determine why the Y955C pol gamma allowed more 8-oxo-dG in DNA and oxidative stress. We performed DNA polymerase reactions with both wild type and mutant polymerases and then developed molecular models with 8-oxo-dG in the active site and the incorporation of different dNTPs. The results indicated that the Y955C pol gamma was more likely to insert the 8-oxo-dG lesion instead of the normal nucleotide dGTP. Once the 8-oxo-dG was present in DNA, the Y955C pol gamma was also more likely to incorporate a dATP opposite this lesion which results in a mutation. The research explains why these PEO patients present phenotypes normally associated with oxidative stress and Parkinson disease.[unreadable] The mitochondrial genome is highly susceptible to damage by reactive oxygen species (ROS) generated endogenously as a by-product of respiration. ROS-induced DNA lesions, including oxidized bases, abasic (AP) sites, and oxidized AP sites, cause DNA strand breaks and are repaired via the base excision repair (BER) pathway in both the nucleus and mitochondria. Repair of damaged bases and AP sites involving 1-nucleotide incorporation, named single-nucleotide (SN)-BER, was observed with mitochondrial and nuclear extracts. However, the repair of oxidized deoxyribose fragments at the 5' terminus after strand break would requires another mode of base excision repair known as Long-Patch base excision repair that was thought to be absent in mitochondria. We showed the presence of a 5' exo/endonuclease in the mitochondrial extracts of mouse and human cells that is involved in the repair of a lyase resistant AP site analog via multinucleotide incorporation that is consistent with a novel long-patch base excision repair mechanism.